1. Field of Invention
The present invention relates generally to fluorescent probes and assays. The probes include a quencher having a moiety that, upon undergoing a change in oxidation state, ceases to act as a quencher of fluorescence. In assays of the invention, the presence of a target substance is detected by the switching of fluorescence mediated by the change in oxidation state of the quencher.
2. Background
There is a continuously expanding need for rapid, highly specific methods of detecting and quantifying chemical, biochemical and biological substances as analytes in research and diagnostic mixtures. Of particular value are methods for measuring small quantities of nucleic acids, peptides, saccharides, pharmaceuticals, metabolites, microorganisms and other materials of diagnostic value. Examples of such materials include narcotics and poisons, drugs administered for therapeutic purposes, hormones, pathogenic microorganisms and viruses, peptides, e.g., antibodies and enzymes, and nucleic acids, particularly those implicated in disease states.
The presence of a particular analyte can often be determined by binding methods that exploit the high degree of specificity that characterizes many biochemical and biological systems. Frequently used methods are based on, for example, antigen-antibody systems, nucleic acid hybridization techniques and protein-ligand systems. In these methods, the existence of a complex of diagnostic value is typically indicated by the presence or absence of an observable “label” which is attached to one or more of the interacting materials. The specific labeling method chosen often dictates the usefulness and versatility of a particular system for detecting an analyte of interest. Preferred labels are inexpensive, safe, and capable of being attached efficiently to a wide variety of chemical, biochemical, and biological materials without significantly altering the important binding characteristics of those materials. The label should give a highly characteristic signal, and should be rarely, and preferably never, found in nature. The label should be stable and detectable in aqueous systems over periods of time ranging up to months. Detection of the label is preferably rapid, sensitive, and reproducible without the need for expensive, specialized facilities or the need for special precautions to protect personnel. Quantification of the label is preferably relatively independent of variables such as temperature and the composition of the mixture to be assayed.
In addition to, or rather than, being directly detected, many dye labels operate to quench the fluorescence of an adjacent second fluorescent label. Because of its dependence on the distance and the magnitude of the interaction between the quencher and the fluorophore, the quenching of a fluorescent species provides a sensitive probe of molecular conformation and binding, as well as other interactions. An exemplary application of fluorescent reporter quencher pairs is found in the detection and analysis of nucleic acids.
Fluorescent nucleic acid probes are important tools for genetic analysis, in both genomic research and development, and in clinical medicine. As information from genome projects accumulate, the level of genetic interrogation mediated by fluorescent probes will expand enormously. One particularly useful class of fluorescent probes includes self-quenching probes, also known as fluorescence energy transfer probes, or FET probes. The design of different probes using this motif may vary in detail. In an exemplary FET probe, both a fluorophore and a quencher are tethered to a nucleic acid. The probe exists in a conformation in which the fluorophore is proximate to the quencher and the probe produces a signal only as a result of its hybridization to an intended target, separating the fluorophore and quencher. Despite the limited availability of FET probes, techniques incorporating their use are rapidly displacing alternative methods.
Fluorescent-based reporter gene assays are also known. The most general reporter systems are those that encode an enzyme that acts upon a small molecule substrate to produce a colorimetric or fluorescent signal. Fluorescence is a significantly more sensitive technique, particularly amongst a background of cellular matter (Naylor, Biochem. Pharmacol., 58: 749-757 (1999)). Pro-fluorescent substrates have been developed for β-lactamase, β-galactosidase, β-glucuronidase, horseradish peroxidase and alkaline phosphatase (Schenborn et al., Mol. Biotechnol., 13: 29-44 (1999)). These enzymes all act upon their substrates to remove an epitope that quenches fluorescence (the first three cleave a glycosidic linkage, horseradish peroxidase oxidizes saturated bonds to conjugated alkenes and alkalinephosphatase hydrolyzes a phosphate ester). These enzymes are commonly applied for both in vitro and in vivo assays. Alkaline phosphatase has been engineered to create a secreted form, eliminating the need to lyse host cells to measure activity (Berger et al., Gene 66: 1-10 (1988)). This is a significant advantage since many cell types possess endogenous phosphatases that can produce high background in the experiment. However, both alkaline phosphatase and horseradish peroxidase are widely used as reporter conjugates in in vitro applications where competing enzymatic activity is excluded.
One of the earliest assay systems designed to monitor cellular transcriptional activity made use of chloramphenicol acetyltransferase (CAT), an enzyme originating from E. coli (Gorman, Mol. Cell. Biol. 2: 1044-1051 (1982)). The assay for the expression of the enzyme involves the substrate 14C-labeled chloramphenicol, which is acetylated by CAT. The amount of radioactive product is quantified after isolation by thin layer chromatography (TLC). The background activity of CAT is negligible in mammalian cells and the threshold for detection of radioactivity is quite low. However, the need for radioactive isotopes and purification of the acetylated chloramphenicol hinder the use of this system due to cost and hazardous waste production. Recently developed fluorescent CAT substrates (Invitrogen) do not obviate the need for time consuming TLC.
Green fluorescent protein (GFP) is a popular reporter gene (Tsien, Annu. Rev. Biochem. 67: 509-544 (1998)). GFP is not an enzyme, but rather a protein that folds to form an intrinsic fluorophore. This is especially useful for protein localization experiments. Since no substrate is needed there is no risk of perturbing the cell by the addition of a potentially toxic small molecule. Another common problem with exogenous substrates is that many are not membrane permeable and so the cell must be ruptured for contact with the enzyme. Efforts to develop new variants of GFP that fluoresce in different colors have been successful, yielding both cyan and yellow fluorescent proteins (Sawano et al., Nucl. Acids Res., 28: E78 (2000)). Current drawbacks to the use of GFP are the length of time required for fluorophore formation and low sensitivity without the signal amplification that arises from enzymatic turnover.
Inducible fluorescent signals are essential components of genomic, biochemical, and immunoassays. Modern drug discovery platforms require rapid identification of interactions between targets and small molecules. Often such assays rely upon enzymatic activity that generates a fluorescent signal. While many fluorescence-based assays are applied in vitro, reporter genes are designed to be observable within a cell or whole organism thus providing key information about protein-protein interactions and gene expression in vivo. Identifying enzyme/substrate pairs with sufficient specificity and sensitivity for these applications is a significant challenge. The present invention provides a strategy to match an enzyme with a panel of pro-fluorescent substrates, thereby creating a system that is readily adaptable to a wide range of environments and diverse fluorescence signals.